Spectroscopic analysis may be used to analyze biological tissues in vivo and in vitro. Recording and analysis of spectral signals from tissue can provide detailed information regarding the physical composition of the target tissue as well as the state of individual physical components. Many molecules may have unique spectral signatures. For example hemoglobin may have distinct spectra depending on its oxygenation. The difference in the spectra between oxy- and deoxy-hemoglobin has been used in multispectral and hyperspectral methods to determine the oxygen content of retinal arteries and veins.
Some described methods and instruments may be limited by the need to manually adjust settings, slow acquisition times, limited field-of-view, poor spatial and/or spectral resolution and insufficient spectral range. In addition, spectral signals, may be subject to deleterious optical effects such as diffraction, absorption, contaminating emissions and scatter by any material in the light path between the illumination source and recording apparatus. Variations in background or surrounding tissue pigmentation can affect the spectroscopic profile of a target since most tissues are semi-transparent. In the case of the retina, recording spectral information may be confounded by the effects of a number of different tissues that encounter the light going into and coming out of the retina. These tissues may include the tear film, cornea, aqueous humor, iris, lens, lens capsule, vitreous, and vitreous debris (red blood cells, white blood cells, cellular debris, vitreous syneresis), choroicapillaris, choroid, Bruch's membrane, and retinal pigment epithelium (RPE). Collectively, one or more of these tissues contribute “spectral noise” to the signal of interest. “Spectral noise” is used herein to mean any spectral information that is not from or due to the target of interest. For example, cataracts, corneal opacities, intraocular inflammation, vitreous hemorrhage, lens dislocation and epiretinal membranes can significantly alter the spectral profile of any light going into or coming out of the retina.
Polarization is a property of electromagnetic waves that describes the orientation of their oscillations. The orientation of the electric fields of electromagnetic waves emanating from a surface may or may not be correlated resulting in various states of polarization. Measurement of these polarization states has provided useful information regarding some biological targets. Analysis of polarization anisotropy in tissue structures has been limited because of the technical limitations in polarimetry. In the eye, the largest retardance is associated with the cornea although the lens and vitreous also contribute. Structures with the most regular microstructures such as the corneal stroma, nerve fiber layer (NFL), Henle's nerve fiber layer, scleral crescent at optic nerve head, lamina cribrosa, rod and cone photoreceptors, Bruch's membrane and sclera are most likely to generate non-depolarizing polarization. The cornea, NFL and Henle's NFL have demonstrated the most prominent polarization properties. Recently the development of commercial polarimeters has made possible the study of tissue anisotropy in the retina. Such polarimeters are incomplete, however, and do not measure all forms of polarization behavior (e.g., depolarization, diattenuation, and retardance).
Despite the advancement noted in polarimetry, limitations still remain. For example, typical polarimeter instruments are commonly limited by the need to manually adjust settings, slow acquisition times, limited field-of-view, and insufficient dynamic range. A relatively small field-of-view and a long acquisition time can necessitate significant effort in image registration and analysis. In addition, polarization of light is also subject to the same sources of noise as described above for spectral imaging. These limitations likely place an upper limit on the clinical sensitivity and specificity of commercial polarimeters and other polarization measurements.
In the medical sciences, diagnostic technologies based on spectral imaging and polarimetry are relatively few and generally not well accepted in the ophthalmology community for multiple reasons. First, these imaging technologies are not well adapted to clinical use because of long image acquisition times, small field-of-view, poor spatial and/or spectral resolution, limited spectral range and incompatibility with other medical equipment. Second, even in cases where images are acquired there may be no reliable method by which to effectively calibrate spectral or polarization data for the sources of noise as described above. Therefore images commonly vary from one exam to the next and the exact source of the spectral or polarization signals is not clear because of the many sources of noise described above. This can limit the clinical utility of the devices as longitudinal follow-up is important in characterizing the progression or regression of a disease process.